Epidemic typhus, one of the most deadly bacterial diseases affecting humans, is caused by the louse-borne pathogen, Rickettsia prowazekii. Due to the high mortality rate of this disease, the lack of an effective vaccine, and the possibility o aerosol dissemination, R. prowazekii is designated as a select agent and has the potential to pose a severe threat to public health and safety. R. prowazekii is an obligate intracellular, parasitic bacterium that only grows within the cytosol of the host cell, unbounded by a vacuolar membrane. This ability to exploit the intracytoplasmic environment and cause serious human disease provides the basis for our continuing studies to elucidate the mechanisms underlying R. prowazekii virulence and how they impact obligate intracellular growth. This proposal builds upon our success in developing new methods to genetically manipulate R. prowazekii and isolate mutants for characterization. In Specific Aim 1 we will employ these techniques to delete a defined subset of genes hypothesized to affect R. prowazekii virulence including enzymes with membranolytic activities, surface-exposed proteins, and protein modifying enzymes. Gene deletions will be generated using proven homologous recombination methods. Mutants will be assessed for gene-specific phenotypes, alterations in growth and morphology, as well as for virulence-associated phenotypes, such as efficient replication in macrophages and the ability to cause fever and weight loss in an animal infection model. These studies will contribute to the identification of new targets for antimicrobial therapy and vaccines. In Specific Aim 2 we will build upon our recent advances to develop and apply new genetic tools fundamentally important for dissecting rickettsial gene function. We have successfully introduced a new recombinant plasmid into R. prowazekii that provides an extrachromosomal platform for recombinant gene expression and allows us to assign phenotypes to specific genes, as well as characterize essential genes. In addition, this platform will be used to develop regulated gene expression systems in R. prowazekii by examining the expression of a fluorescent protein placed under the control of a variety of potential control elements. Our proven ability to overcome the roadblocks in the genetic manipulation of R. prowazekii and the development of new detection and isolation techniques will accelerate our gene-by-gene dissection of systems that dictate rickettsial virulence and intracellular growth.